An amadoriase oxidizes iminodiacetic acid or a derivative thereof (also referred to as “Amadori compound”) in the presence of oxygen to catalyze a reaction to generate glyoxylic acid or α-ketoaldehyde, amino acid or peptide, and hydrogen peroxide.
Amadoriases have been found in bacteria, yeast, and fungi (e.g., see Patent Documents 1 to 4). Amadoriases have been purified from the genera Aspergillus, Penicillium, Fusarium, Pichia, Coniochaeta, Eupenicillium, Pyrenochaeta, Arthriniuin, Neocosinospora, Corynebacterium and Agrobacterium to determine the amino acid sequence of each amadoriase (e.g., see Non-Patent Documents 1 to 4 and Patent Documents 5 to 9).
These amadoriases can be classified into two types of prokaryotic and eukaryotic amadoriases. The prokaryotic amadoriases derived from procaryotes and the eukaryotic amadoriases derived from eucaryotes have amino acid sequences having high homologies with the amadoriases only in the same type, respectively, whereas the amino acid sequences have extremely low homologies between the different types of the eukaryotic and prokaryotic amadoriases.
The prokaryotic amadoriases had a problem of unfortunately losing their enzyme activities by separating some coenzymes during purification or storage of enzymes because of forming bonds with the coenzymes that are not covalent bonds. In contrast, the aforementioned problem confirmed in the prokaryotic amadoriases is not seen in the eukaryotic amadoriases because the eukaryotic amadoriases form covalent bonds with the coenzymes, and thus the eukaryotic amadoriases have excellent practical characteristics.
In the field of clinical diagnosis of diabetes mellitus, attention has been given to glycated hemoglobin (HbA1c) as a glycemic control marker significant for diagnosis of diabetic patients and control of conditions. For a method of quickly and simply measuring the HbA1c, there has been proposed an enzymic method using an amadoriase, that is, a method of measuring glycated amino acids or glycated peptides, released by decomposing the HbA1c by, e.g., protease (for example, see Patent Documents 10 to 13).
Thermal stability is demanded as an enzymatic property, where an amadoriase as an enzyme for clinical diagnosis of diabetes mellitus is formulated for a kit reagent. A eukaryotic amadoriase derived from a strain of Aspergillus terreus GP1 has exhibited a residual activity of about 40% in heat treatment at 45° C. for 10 minutes (for example, see Non-Patent Document 2). A eukaryotic amadoriase derived from a strain of Fusarium oxysporum S-1F4 has exhibited a residual activity of about 10% in heat treatment at 45° C. for 5 minutes (for example, see Non-Patent Document 5). A eukaryotic amadoriase derived from a strain of Coniochaetidium savoryi ATCC36547 has also exhibited a residual activity of 80% in heat treatment at equal to or less than 37° C. for 30 minutes (for example, see Patent Document 14). Furthermore, each of eukaryotic amadoriases derived from strains of Arthrinium sp. TO6, Pyrenochaeta sp. YH807, Leptosphaeria nodorum NBRC7480, Pleospora herbarum NBRC32012 and Ophiobolus herpotrichus NBRC6158 has exhibited a residual activity of 80% in heat treatment at equal to or less than 40° C. for 30 minutes. A eukaryotic amadoriase derived from a strain of Neocosmospora vasinfecta NBRC7590 has also exhibited a residual activity of 80% in heat treatment at equal to or less than 45° C. for 30 minutes. A eukaryotic amadoriase derived from a strain of Curvularia clavata YH923 has exhibited a residual activity of 80% in heat treatment at equal to or less than 50° C. for 30 minutes (for example, see Patent Document 14).
However, further thermal stability is needed where these eukaryotic amadoriases are used as enzymes for clinical diagnosis. That is, further higher thermal stability is demanded in consideration of formulation of the eukaryotic amadoriases, as enzymes for clinical diagnosis of diabetes mellitus, for a kit reagent, and use as enzyme sensors, although the enzyme derived from the strain of Curvularia clavata YH923, having the highest thermal stability, has exhibited a residual activity of 80% in heat treatment at equal to or less than 50° C. for 30 minutes.
For a general technique, there has been known a method of adding mutations to DNAs encoding enzymes, introducing substitutions into the amino acids of enzymes and selecting enzymes with excellent thermal stability in order to improve the thermal stability of the enzymes. In addition, if an example of improving thermal stability by amino acid substitution in enzymes with high homology has been already known, improvement in the thermal stability can be expected based on this information.
Indeed, in a prokaryotic amadoriase derived from Corynebacterium bacteria, the thermal stability of the prokaryotic amadoriase has been demonstrated to be improved by replacing several amino acids (for example, see Non-Patent Document 5), and thermal stability can be also introduced into other prokaryotic amadoriases.
However, since, as described above, the amino acid sequences of the amadoriases have extremely low homologies between the types of the eukaryotic amadoriase and the prokaryotic amadoriase, it was impossible to expect the improvement of the thermal stability of the eukaryotic amadoriase on the basis of information on amino acid mutations involved in the thermal stability of the prokaryotic amadoriase derived from Corynebacterium bacteria.
Also, there has been no report that the well-known eukaryotic amadoriase was improved in thermal stability by replacement of amino acids, and existing information on the thermal stability of the eukaryotic amadoriase can not be utilized. Extensive, specific researches are demanded for determining which amino acid to replace in a sequence in order to practically improve the eukaryotic amadoriase type in thermal stability.
Patent Document 1: Japanese Patent Publication No. 05-33997;
Patent Document 2: Japanese Patent Application Laid-Open Publication No. 2000-270855;
Patent Document 3: Japanese Patent Application Laid-Open Publication No. 07-289253;
Patent Document 4: Japanese Patent Application Laid-Open Publication No. 08-336386;
Patent Document 5: Japanese Patent Application Laid-Open Publication No. 2003-235585;
Patent Document 6: Japanese Patent Application Laid-Open Publication No. 2004-275063;
Patent Document 7: Pamphlet of WO 2004/104203;
Patent Document 8: Japanese Patent Application Laid-Open Publication No. 11-155579;
Patent Document 9: Japanese Patent Application Laid-Open Publication No. 2003-79386;
Patent Document 10: Japanese Patent Application Laid-Open Publication No. 2001-95598;
Patent Document 11: Bulletin of Japanese Patent Publication No. 05-33997;
Patent Document 12: Japanese Patent Application Laid-Open Publication No. 11-127895;
Patent Document 13: Pamphlet of WO 97/13872;
Patent Document 14: Japanese Patent Application Laid-Open Publication No. 2004-275013;
Non-Patent Document 1: Arch. Microbiol. 178, 344-50, 2002;
Non-Patent Document 2: Eur. J. Biochem. 242, 499-505, 1996;
Non-Patent Document 3: Mar. Biotechnol. 6, 625-32, 2004;
Non-Patent Document 4: Biosci. Biotechnol. Biochem. 59, 487-91, 1995;
Non-Patent Document 5: Appl. Environ. Microbiol. 69, 139-45, 2003.